Production of hepatitis A virus in vitro utilizing a persistently virus infected cell culture system

ABSTRACT

The present invention relates to a method for producing human hepatitis A virus in vitro employing tissue culture techniques. In particular, the present invention relates to an in vitro tissue culture procedure utilizing a persistently infected cell line that produces high titers of hepatitis A virus. The hepatitis A virus thus produced is a source of hepatitis A virus antigens.

This application is a continuation of application Ser. No. 344,683,filed 2/1/82 now abandoned.

BACKGROUND OF THE INVENTION

Various tissue culture procedures for propagating hepatitis A virus havebeen disclosed, such as, for example, U.S. Pat. No. 4,164,566; EuropeanPatent Application No. 25,745; Frosner, et al, Propagation of HumanHepatitis A virus in a Hepatoma Cell Line, Infection, 6: 303-306;Flehmig, Hepatitis A-virus in cell culture: I. Propagation of DifferentHepatitis Virus Isolates in a Fetal Rhesus Monkey Kidney Cell Line(Rfnk-4), Med. Microbiol. Immunol., 168: 239-248; and Daemer, et al,Propagation of Human Hepatitis A Virus in African Green Monkey KidneyCell Culture: Primary and Serial Passage, Infection and Immunity, 32:388-393. However, such methods are hereinafter referred to as "acuteinfection" procedures wherein multiple passages of cell free virus intissue cultures are required before an acceptable virus titer isobtained. In accordance with acute infection procedures, a cell tissueculture is infected with a specimen containing hepatitis A virus. Theinfected cell culture is grown in an appropriate medium through"confluency" and then the fully grown cells are allowed to "age". The"aged" cells are " killed" and the virus is extracted. The extractedcell free virus is subsequently utilized to reinfect a new cell culture.This process is repeated several times, generally referred to aspassages of the virus, until an acceptable virus titer is obtained.However, "acute infection" or so-called "reinfection" procedures aregenerally time consuming and inefficient in that such proceduresgenerally involve termination of the infected cultures and infection ofnew cultures in order to obtain a suitable virus titer. As a result,material, manpower, and physical space requirements for tissue cultureprocedures utilizing acute infection techniques are high. Furthermore,some disclosed acute infection procedures are "indirect", that is,require repeated adaptation of the virus in marmosets before propagationin tissue cultures.

It is an object of the present invention to provide a tissue cultureprocedure for the production of hepatitis A virus in vitro, whicheliminates the tedious and time consuming acute infection proceduresrequiring "reinfection" or repeated passages of cell free virus. Also,it is a further object of the present invention to provide a procedurefor increasing the yields of hepatitis A virus obtained from a tissueculture procedure.

SUMMARY OF THE INVENTION

The present invention relates to a method for production of humanhepatitis A virus in an in vitro cell culture system comprisingmaintaining a persistently infected virus producing cell line andharvesting hepatitis A virus from the persistently infected virusproducing cell line. The present invention also relates to a method forestablishing a persistently infected human hepatitis A virus producingcell line, said method comprising: (a) infecting a suitable cell culturewith human hepatitis A virus and (b) maintaining the infected cellculture at log phase growth until the cell culture is capable ofproducing a stable virus titer. The present invention further relates toa method of increasing the yield of human hepatitis A virus obtainedfrom an infected cell line, said method comprising harvesting the humanhepatitis A virus from the infected cell line utilizing anonphysiological mixture comprising a chelating agent and a nonionicdetergent in a buffered medium.

The hepatitis A virus obtained as a result of the methods of the presentinvention is a source of viral antigens which are useful in diagnosticprocedures for the detection of type A hepatitis.

DETAILED DESCRIPTION OF THE INVENTION

According to the present invention, hepatitis A virus is propagated inan in vitro cell culture system wherein said cell culture systemcomprises a persistently infected virus producing cell line. As usedherein, the term "persistently infected virus producing cell line"refers to a virus infected cell line maintained at log phase growth andcapable of producing a stable virus titer. To establish a persistentlyinfected virus producing cell line, a suitable cell line is infected bymeans of a single treatment with an inoculum obtained from a humanclinical specimen containing the virus, and the infected cell line isthereinafter maintained at log phase growth until essentially all thecells in the culture become infected and a stable virus titer isachieved. The term "maintained at log phase growth" refers to a processwherein infected cells of a culture are grown in a nutrient media untilthe cells reach confluency, generally within seven to ten days andthereafter upon reaching confluency and prior to "aging", the liveinfected cells are repeatedly subcultured, that is, a portion of theinfected cell culture is transferred to a fresh culture vessel and againallowed to grow in a nutrient medium to confluency. The term, "stablevirus titer", as used herein, refers to a cell culture system whereinessentially all of the cells in the culture are infected with the virusand each infected cell is producing large numbers of the virus.Generally, it has been found that a stable virus titer is obtainedwithin four to five subcultures following the initial inoculation. Oncea stable virus titer is achieved, that is, a persistently infected virusproducing cell line is established, each succeeding subculture of theinfected cell line thereafter becomes a persistently infected virusproducing cell line capable of immediately producing a high virus titer.It should be noted that in the method of the present invention at notime prior to harvesting of the virus is it necessary that the infectedcells be lysed, i.e., killed, and the resulting cell free virus utilizedto reinfect a fresh culture, as required with acute infectiontechniques.

The term "suitable cell line" as used herein refers to a cell linecapable of becoming persistently and stably infected, i.e., essentiallyevery cell becomes infected, remains infected throughout its life cycleand transmits infection to its progency cells.

Illustrative of a "suitable cell line" effective in the method of thepresent invention include human liver tumor lines PLC/PRF/5 (hereinafterreferred to as "Alexander") and Hep. G2 (hereinafter referred to as"G2") described in Br. J. Cancer (1976) 34, 509 and Nature (1979), Vol.282, 615, respectively. It is preferred to employ the human liver tumorline "Alexander" in the methods of the present invention.

Subculturing of the infected cell culture is generally carried oututilizing conventional subculturing procedures, i.e., a trypsinprocedure described in, for example, Earle W. R., Tissue Culture, CowdryE. V. ed. Laboratory Technique in Biology and Medicine, 2nd Ed. (1948).

The intracellar virus produced utilizing the persistently infected virusproducing cell line of the present invention, may be harvested inaccordance with known techniques employing conventional procedures andreagents to lyse and cells containing the virus. The preferred methodfor harvesting the hepatitis A virus is to first allow the infectedcells of the persistently infected virus producing cell line uponreaching confluency to age for approximately two to ten days andpreferably seven days. Following this "aging" period, the growth mediais removed from the culture and the cells are lysed. The cell lysatesare cleared of nuclei, cell organelles, and debris by centrifugation toyield hepatitis A virus. It has been found that the yields of virus areunexpectedly and significantly increased when the virus infected cellsare lysed utilizing a nonphysiological solution comprising a chelatingagent and a nonionic detergent in a buffered solution. The chelatingagents and nonionic detergents employed in the methods of the presentinvention are readily ascertained by one of ordinary skill in the artand include for example, chelating agents such as sodiumethylenediaminetetracetate (EDTA) and nonionic detergents including, forexample, polyethylene glycol p-isooctylphenyl ethers such as Triton X-100(poly(oxy-1,2-ethanediyl),alpha-[4-(1,1,3,3-tetramethylbutyl)phenyl]-.omega.-hydroxy-(octoxynol9)). The hepatatis A virus thus obtained possesses the necessaryproperties for the preparation of an antigen employed in diagnosticimmunoassays.

The nutrient medium employed in the methods of the present invention isa medium sufficient to maintain the cells at temperatures permittingpropagation of both the cells and the virus in the cell culture.Generally, such temperatures range from about 30° to 39° C. The nutrientgrowth medium may be, for example, Eagle's minimal essential medium,Delbecco's minimal essential medium RPM1-1640, Eagle's medium with HEPESbuffer and L-15-a phosphate buffered medium. It is preferred to employ aphosphate or HEPES buffered medium. In addition, the growth media isgenerally supplemented with a mixture comprising from 10-30% fetal calfserum, 100 μg/ml of penicillin and 100 units of streptomycin. Inaddition, the pH of the persistently infected virus producing cell lineis generally that of the medium employed, approximately a pH of 7.6.Furthermore, the buffered solution utilized to lyse the cells isgenerally at the same pH as the growth medium. It is preferred that thepH of the buffered solution be approximately 7.6.

The following illustrative, nonlimiting examples will serve to furtherdemonstrate to those skilled in the art the present invention.

EXAMPLE I Establishment of a Persistently Infected Virus Producing CellLine

The hepatitis A virus inoculum employed in the examples was obtainedfrom a human clinical fecal specimen that was clarified, filtered anddiluted 1:10 (w/v) in phosphate-buffered saline. Prior to infection, anAlexander human liver tumor cell line was grown in a nutrient mediumsupplemented with 10% fetal calf serum, 100 μg/ml of penicillin and 100units of streptomycin to mid-log phase (40-60% confluency) in 60 mmpetri dishes. The cultures were then drained of the nutrient medium andtreated with 2 ml of DEAE-dextran in unsupplemented media at 25 μg/ml at37° C. for 20 minutes. To the DEAE-dextran treated cultures was added 4ml of the filtered inoculum in complete medium at a 1:50 dilution. Anuninfected control culture was similarly treated, except for infectionwith the inoculum. The medium was drained and replaced the next day. Theinfected and uninfected control cultures were subcultured approximatelyevery seven days. The cultures were periodically monitored for evidenceof hepatitis A virus replication by two types of immunoassays. Indirectimmunofluorescence (IMF) assays were carried out on coverslip culturesusing human immunoglobulins (IgG purified from a clinical sample ofhuman serum) and fluorescein-conjugated goat antihuman IgG. This assayidentified virus-producing cells by fluorescence-labeling. Utilizing theIMF techniques with respect to the infected cultures, fluorescent cellsindicating infection, appeared as early as two to three weekspost-infection. At first, only 1%-2% cells in the cultures werefluorescent. On continued subculturing, a higher percentage of the cellswere fluorescent, indicating increasing amounts of virus infected cells.The control cultures exhibited no fluorescence. Viral replication wasalso monitored by a solid-phase radioimmunoassay (RIA). The cells werelysed in phosphate buffered saline by repeated freezing and thawing. Thecell extracts were clarified and then tested for viral antigen in anassay involving anti-HAV IgG-coated beads and ¹²⁵ I-labeled anti-HAVantibodies. The sensitivity and the results obtained as a result of theradioimmunoassay procedure were consistent with the results obtainedfrom the IMF assays.

A persistently infected virus producing cell line was established withinthree months following infection; that is, the tissue cultures becameessentially 100% infected and, thereafter upon subsequent subculturing,continuously produced high stable titers of hepatitis A virus.

Properties of the Persistently Infected Virus Producing Cell Line

The above-described persistently infected virus producing cell line wassubcultured forty times over a period of eleven months. The followingsummarizes the characteristic properties of the persistently infectedvirus producing cell line thus established:

At subculture 2 (week 3), 2% of the cells become infected and wereproducing virus detectable by RIA and IMF assays. Following the sixthsubculture, approximately week 7, 40-50% of the cells became infected.Following the ninth subculture and all subsequent subcultures, the virusproducing cell line system had become positively infected. There wasrapid horizontal spread of the virus ensuring the infection of everycell within the culture. Furthermore, on cell division, there must havebeen a consistent transmission of the virus to progency cells.

It has been noted that the established persistently infected virusproducing cell line system did not require a special medium. Eagle'sminimal essential medium, Delbecco's minimal essential, RPM1-1640,Eagle's medium with HEPES buffer and L-15 have been successfullyemployed. The cells of the virus-cell system have the same morphologicaland growth characteristics as the parent Alexander culture. The cellsgrow in monolayers under either stationary or dynamic conditions.

During the log-phase growth, cells of the persistently infected virusproducing cell line system undergo cell division in about ten to sixteenhours. At a split ratio of 1:10, a subculture reaches confluency inseven to nine days, saturating at a cell density of 8×10⁴ cells per cm².Confluent cultures could be held and allowed to age in a healthy stateby changing to a maintenance medium containing 1% fetal calf seruminstead of 10%. By changing with fresh maintenance medium every threedays, it has been noted that the cultures could be held for more thantwo months.

To illustrate the hardiness of the parent cell line, infected Alexandercultures were left at room temperature without being fed fresh media fortwo weeks. The cells aged and many of the cells lysed and died whileothers exhibited severe pathological changes such as vacuolization,granulation, refractility, and long cytoplasmic processes. The cultureswere fed maintenance medium and left unattended for more than twomonths. In spite of such neglect (harsh suboptimal conditions) thesecells could be nurtured back to robust growth after several subculturingusing proper care.

EXAMPLE II Harvesting of the Virus

To harvest the virus, a subculture of the persistently infected virusproducing cell line established in Example I is drained of nutrientmedia and washed with a solution of 1 mM EDTA in phosphate bufferedsaline. Following washing, the infected cells are detached and lysedsimultaneously with a solution comprising 1 mM EDTA and 0.5% TritonX-100 in phosphate buffered saline. The lysed cells are incubated atroom temperature for approximately 10-15 minutes. The cell lysates arecentrifuged to clear nuclei, cell organelles and other debris to yield aclarified cell extract containing the hepatitis A virus.

Characterization of the Virus

The persistently infected virus producing cell system established inExample 1, replicates a virus having biophysical, morphological,immunological, and biological properties characteristic of hepatitis Avirus isolated from human sources. The virus obtained as a result of themethods of the present invention banded with a bouyant density of CsClof about 1.34 g/cc and a sedimentation velocity of about 160s insucrose. The virus appeared as naked nucleocapsids, having a diameter of27 nm, that can be specifically bound by human anti-hepatitis A virusantibodies in solid phase RIA. Upon inoculation onto fresh Alexandercells, the virus replicated to high titers. Upon inoculation intomarmosets, the virus induced, within one week, clinical sysmptoms ofhepatitis confirmable by serum glutamic pyruvic transaminase elevationand presence of hepatitis A virus antigens in fecal and liver extracts.

EXAMPLE III

The persistently infected virus producing cell line described hereinreadily adopted to mass virus propagation. In general, a large-scalescheme for producing hepatitis A virus utilizing a persistently infectedvirus producing cell line involves the maintenance of two lines ofcultures: "stock": cultures and "production" cultures. Stock culturesprovide infected cells to seed large numbers of production cultureswhich are intended for virus harvests, as well as a small number (1/10)of stock cultures for replenishment. Stock cultures are kept in the mostvigorous state of growth. The stock cultures are seeded lightly (5×10⁷cells per 6000 cm² cell factory) so that they reach confluency inapproximately one week and are subcultured immediately. These cells areused to set up both stock cultures and production cultures. The cellsare preferably subcultured at "late log phase", thereby permitting thecells to immediately divide without a lag period. It is important toexamine the cultures microscopically on days one and two to confirm thatthe cells immediately divide. Seeding with aged, unhealthy, or damagedcells may result in a long lag period of three to seven days before thecells recover and divide, thereby delaying virus harvesting. Productioncultures are seeded at a higher density (10⁸ cells per cell factory) inorder to allow the cultures to reach confluency earlier generally dayfive to seven. The production cultures are fed maintenance medium andupon reaching confluency are allowed to "age" for several days. Timecourse studies have indicated that the virus yield more than doublesduring the "aging" period. The production cultures are sacrificed andharvested for virus generally between day ten to fourteen. A singlestock culture generally provides in one week approximately enough cellsfor one stock and nine production cultures. Within one week, the nineproduction cultures yield on the average approximately 5×10⁸ cells, 10¹²-10¹³ infectious virus particles or 5×10⁸ equivalent of antigen asmeasured using a standard radioimmunoassay procedure, employing Abbott'sHAVAB RIA diagnostic kit and reagents.

The following describes a representative large-scale system:

Cell Culturing

An appropriate number of virus infected cells at late log phase growthand obtained from the persistently infected virus producing cell lineestablished in Example 1 and are suspended in 1.8 l of complete medium(L-15 supplemented with 100 units per ml of penicillin, 100 μg/mlstreptomycin and 10% fetal calf serum). The cell suspension isintroduced by gravity flow into a cell factory (NUNC, 6000 cm²) evenlydistributing the cells among the ten layers. The seeding cell number forstock cultures is 5×10⁷ and for production cultures, is 10⁸. The cellfactory is kept at 37° C. and periodically microscopically examined tomonitor cell growth.

Cell Subculturing

Stock cultures are subcultured utilizing a conventional trypsinprocedure. The cell factory is drained of media, rinsed with 1 l ofphosphate buffered saline and incubated with 0.8 l of 0.025% trypsinuntil cells lift. The cells are then collected. All operations arecarried out by gravity flow at room temperature under sterileconditions. To the collected cells is added 40 ml of fetal calf serumand resulting cell suspension is centrifuged. The resulting cell pelletis gently resuspended in 50 ml complete medium. Cell yield is determinedin a hemocytomer.

Virus Harvesting

Production cultures, upon reaching confluency generally on days five toseven, are drained of 90% of the medium and replaced with 90% freshmaintenance medium (same as previously employed except fetal calf serumis lowered to 2%). The cultures are incubated for an additional three toseven days. The cultures are drained, rinsed with 1.5 l of phosphatebuffered saline and the cells are lysed with a mixture of 0.5 lphosphate buffered saline, 1 mM EDTA and 0.5% Triton X-100. The celllysates are collected, and then clarified by centrifugation at 2000xgfor ten minutes, and at 10,000xg for fifteen minutes at 4° C. The virusyield obtained is routinely measured by RIA.

EXAMPLE IV Preparation of Hepatitis A Virus Antigen

Hepatitis A viral antigen is prepared by inactivating the tissue culturederived virus by conventional formalin treatment procedures. Generally,the virus extracted from the infected cells is incubated with a 1:4000dilution of a 37% formaldehyde solution at 37° C. for three days and theresulting hepatitis A viral antigen is then collected.

As evidenced from the above Examples, the persistently infectedhepatitis A virus producing cell line established in accordance with themethods of the present invention is effective in producing high titersof hepatitis A virus and hepatitis A viral antigen. It should be noted,that the number of subcultures necessary to establish a persistentlyinfected virus producing cell line, following the initial inoculation ofthe cell line, is readily ascertained by one of ordinary skill in theart and will vary depending on factors such as, the source of inoculum,specific type of cell line and the like. In addition, other propertiesof the cell culture such as, for example, the number of cell divisionsof the cell line is capable of, life-span of the cell line, rate ofgrowth of the cell line, i.e., the time required for the cells todivide, number of subcultures that may be derived from the infected cellline, and the like, are readily ascertained, depending on the specifictype of cell line initially infected in the procedures described above.Another advantage to the system described in the preceding Examples isthe fact that carbon dioxide incubators are not required. In addition tothe cell factory employed in Example 3, other suitable growth vessels,such as roller bottles have been successfully employed.

As previously mentioned, although other methods for lysing the virusfrom the infected cells may be employed, such as freeze-thawingtechniques and physiological lysing solutions such as phosphate bufferedsaline, it is preferred to employ a nonphysiological solution comprisinga chelating agent and a nonionic detergent in a buffered solution. It ispreferred that phosphate buffered saline having a pH of about 7.6 beemployed as the buffered solution. It has been found that the yields ofhepatitis A viral antigen produced utilizing the chelating agent and thenonionic detergent to lyse the virus is approximately eight to ninetimes as great as the yields of hepatitis A viral antigen obtainedutilizing lysing solutions that do not contain a nonionic detergent.

Although this invention has been described with respect to specificmodifications, the details thereof are not to be construed aslimitations, for it will be apparent that various equivalents, changesand modifications may be resorted to without departing from the spiritand scope thereof and it is understood that such equivalent embodimentsare intended to be included therein.

What is claimed is:
 1. A tissue culture method for propagating hepatitisA virus in a hepatitis A virus-infected cell culture comprising thesteps of:(a) infecting a susceptible cell culture with a hepatitis Avirus; (b) growing the infected cells in the culture in a nutrient mediauntil the cell culture reaches confluency; (c) transferring some of thelive, infected cells to a fresh nutrient media wherein the cells aremaintained in log phase growth; (d) repeating steps (b) and (c) untilessentially all of the cells in the culture become infected and apersistently-infected hepatitis A virus-producing cell line isestablished; and, (e) harvesting the hepatitis A virus.
 2. A methodaccording to claim 1 wherein the hepatitis A virus is harvestedutilizing a nonphysiological mixture of a chelating agent and a nonionicdetergent in a buffered solution to lyse the infected cells.
 3. A methodaccording to claim 2 wherein the nonphysiological mixture comprisessodium ethylenediaminetetraacetate and a polyethylene glycolp-isoctylphenyl ether in a buffered solution.
 4. A method according toclaim 3 wherein the polyethylene glycol p-isooctylphenyl ester ispoly(oxy-1,2-ethanediyl),alpha-[4-(1,1,3,3-tetramethylbutyl)phenyl]-.omega.-hydroxy-(octoxynol9).
 5. A method according to claim 1 wherein the susceptible cellculture is infected by treating the cell culture with an inoculumobtained from a human clinical specimen containing hepatitis A virus. 6.A method according to claim 1 wherein the infected cell culture isobtained by treating a suitable cell culture with infected cellscontaining hepatitis A virus, wherein such infected cells are obtainedfrom a persistently infected hepatitis A virus producing cell line.
 7. Amethod according to claim 1 wherein the susceptible cell culture is anAlexander or G2 human liver tumor cell line.
 8. A method according toclaim 7 wherein the susceptible cell line is Alexander human liver tumorcell line.
 9. A method according to claim 8 wherein the hepatitis Avirus is harvested utilizing a nonphysiological mixture comprising achelating agent and a nonionic detergent in a buffered solution to lysethe infected cells.
 10. A method according to claim 9 wherein thenonphysiological mixture comprises sodium ethylenediaminetetraacetateand a polyethylene glycol p-isooctylphenylether in a buffered solution.11. A method according to claim 1 wherein the cell culture, followingstep (a), reaches confluency within seven to ten days.
 12. A methodaccording to claim 1 wherein steps (b) and (c) are repeated from four toseven times before a persistently infected hepatitis A virus producingcell line is established.
 13. A method according to claim 12 whereinsteps (b) and (c) are repeated from four to five times before a stablepersistently infected hepatitis A virus producing cell line isestablished.
 14. A tissue culture method for establishing apersistently-infected hepatitis A virus-producing line, comprising thesteps of:(a) infecting a susceptible cell culture with a hepatitis Avirus; (b) maintaining the live, infected cells in the culture in anutrient media until the cell culture reaches confluency; (c)transferring some of the live, infected cells to fresh nutrient mediawherein the cells are maintained in log phase growth; and, (d) repeatingsteps (b) and (c) until essentially all of the cells in the culturebecome infected and a persistently-infected hepatitis A virus-producingcell line is established.
 15. A method according to claim 14 wherein thesusceptible cell culture is infected by treating the cell culture withan inoculum obtained from a human clinical specimen containing hepatitisA virus.
 16. A method according to claim 15 wherein the susceptible cellculture is an Alexander or G2 human liver tumor cell line.
 17. A methodaccording to claim 16 wherein the susceptible cell culture is Alexanderhuman liver tumor cell line.
 18. A method of growing hepatitis A virusin vitro comprising the steps of:(a) growing cells infected withhepatitis A virus in a susceptible cell culture in a nutrient media atlog phase growth; (b) transferring some of the live, infected cells tofresh nutrient media wherein the cells are maintained at log phasegrowth; and, (c) repeating steps (a) and (b) until essentially all ofthe cells in the culture have become infected to produce apersistently-infected hepatitis A virus-producing cell line.
 19. Amethod of growing hepatitis A virus in vitro comprising the steps of:(a)growing cells infected with hepatitis A virus in a susceptible cellculture in a nutrient media at log phase growth; (b) transferring someof the live, infected cells to fresh nutrient media wherein the cellsare maintained at log phase growth; (c) repeating steps (a) and (b)until essentially all of the cells in the culture have become infectedto produce a persistently-infected hepatitis A virus-producing cellline; and (d) harvesting the hepatitis A virus.